Peptide adjuvants

ABSTRACT

Adjuvant compositions comprising specific 5mer polypeptides in combination with antigen delivery systems and/or immunostimulatory molecules, such as immunostimulatory nucleic acid sequences, for enhancing the immune response of a coadministered antigen, are described.

PRIOR APPLICATION INFORMATION

The instant application claims the benefit of U.S. Provisional PatentApplication 61/118,533, filed Nov. 28, 2008.

FIELD OF THE INVENTION

The present invention relates generally to adjuvants, immunogeniccompositions, and methods useful for polynucleotide-based vaccination.The present invention provides compositions and methods useful forenhancing immune response, especially the humoral immune response ofvertebrates

BACKGROUND OF THE INVENTION

Vaccine compositions often include immunological adjuvants to enhanceimmune responses. For example, Complete Freund's adjuvant (CFA) is apowerful immunostimulatory agent that has been successfully used withmany antigens on an experimental basis. CFA includes three components: amineral oil, an emulsifying agent, and killed mycobacteria, such asMycobacterium tuberculosis. Aqueous antigen solutions are mixed withthese components to create a water-in-oil emulsion. Although effectiveas an adjuvant, CFA causes severe side-effects, including pain, abscessformation and fever, primarily due to the presence of the mycobacterialcomponent. CFA, therefore, is not used in human and veterinary vaccines.

Immunologic adjuvants help increase immune responses induced byvaccines. Different mechanisms have been proposed to explain theenhanced antigen specific immune response generated by adjuvantedvaccine. First, adjuvant can promote a slow release of the antigenexposing it to the immune system for a longer period of time andconsequently stimulating a stronger and possibly better defined immuneresponse. Second, adjuvant can also help delivery and uptake of theantigenic complex to antigen presenting cells (APCs) such as macrophagesand dendritic cells which in turn can migrate to lymphoid organs andinitiate a concerted response in interaction with T and B cells. Third,immune cells including APCs can be directly activated by adjuvant andthen initiate a faster and stronger immune response through thesubsequent stimulation of T and B cells. Oil-in-water emulsion ingestedby macrophage which then can migrate to draining lymph nodes, or TLRsstimulating molecules such as unmethylated CpG dinucleotide-containingDNA, are examples of adjuvant acting mainly according to thesemechanisms. An interesting paradigm regarding immune reaction is thatimmune responses are generally more robust when stimulated by an antigenof rare occurrence than by an antigen frequently encountered in nature.The present study explores the possibility of using short peptidicsequences not present or observed only once in known proteomes asimmunomodulators to enhance vaccine-induced immune responses andprotection against lethal viral infections.

SUMMARY OF THE INVENTION

According to a first aspect of the invention, there is provided a methodof stimulating an immune response to an antigen comprisingco-administering to an individual in need of such treatment an effectiveamount of a composition comprising an antigen and a peptide comprisingat least one amino acid sequence as set forth in table 1.

According to a second aspect of the invention, there is provided animmuno-stimulatory peptide sequence consisting of a peptide as set forthin table 1.

According to a further aspect of the invention, there is provided acomposition comprising an effective amount of an adjuvant peptidecomprising an amino acid sequence selected from the group consisting of:KWCEC (SEQ ID No. 4); KYMCW (SEQ ID No. 12); CYWWW (SEQ ID No. 14);EHWCM (SEQ ID No. 15); FCCWW (SEQ ID No. 16); TCCMW (SEQ ID No. 17);TCWWH (SEQ ID No. 18); TCYWW (SEQ ID No. 19); WMICM (SEQ ID No. 20) andYWHMW (SEQ ID No. 21) and an antigen of interest.

According to another aspect of the invention, there is provided a methodof stimulating an immune response or enhancing an immune response to anantigen comprising administering to an individual in need of or desirousof such treatment an effective amount of an adjuvant peptide comprisingan amino acid sequence selected from the group consisting of: KWCEC (SEQID No. 4); KYMCW (SEQ ID No. 12); CYWWW (SEQ ID No. 14); EHWCM (SEQ IDNo. 15); FCCWW (SEQ ID No. 16); TCCMW (SEQ ID No. 17); TCWWH (SEQ ID No.18); TCYWW (SEQ ID No. 19); WMICM (SEQ ID No. 20) and YWHMW (SEQ ID No.21).

According to another aspect of the invention, there is provided the useof an adjuvant peptide comprising an amino acid sequence selected fromthe group consisting of: KWCEC (SEQ ID No. 4); KYMCW (SEQ ID No. 12);CYWWW (SEQ ID No. 14); EHWCM (SEQ ID No. 15); FCCWW (SEQ ID No. 16);TCCMW (SEQ ID No. 17); TCWWH (SEQ ID No. 18); TCYWW (SEQ ID No. 19);WMICM (SEQ ID No. 20) and YWHMW (SEQ ID No. 21) for stimulating animmune response or enhancing an immune response to an antigen in anindividual in need of or desirous of such treatment

According to a further aspect of the invention, there is provided amethod of preparing a medicament for stimulating an immune response orenhancing an immune response to an antigen comprising mixing aneffective amount of an adjuvant peptide comprising an amino acidsequence selected from the group consisting of: KWCEC (SEQ ID No. 4);KYMCW (SEQ ID No. 12); CYWWW (SEQ ID No. 14); EHWCM (SEQ ID No. 15);FCCWW (SEQ ID No. 16); TCCMW (SEQ ID No. 17); TCWWH (SEQ ID No. 18);TCYWW (SEQ ID No. 19); WMICM (SEQ ID No. 20) and YWHMW (SEQ ID No. 21)with a suitable excipient.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Enzyme-linked immunosorbent spot (ELISPOT) T cell responsefollowing immunization. BALB/c mice were vaccinated I.M. with 50 μg ofpCAGα-HA DNA vaccine containing either: 5mer1 (CHKWD (SEQ ID No. 1)),5mer2 (WHKCE (SEQ ID No. 2)), 5mer3 (CKWRC (SEQ ID No. 3)), 5mer4 (KWCEC(SEQ ID No. 4)), 5mer5 (DCWMD (SEQ ID No. 5)), 9mer1 (CWKCWCMFE (SEQ IDNo. 6)), 9mer3 (WNWCMHWDC (SEQ ID No. 7)), 9mer4 (WHWCMMCWD (SEQ ID No.8)), 13mer1 (HEHWCMMWHCCMI (SEQ ID No. 9)), 13mer3 (HMMCHWMCWCDMH (SEQID No. 10)), or 13mer4 (CHMMCHWMWCCMD (SEQ ID No. 11)) foreign peptidefused to the carboxyl-terminal end of HA. pCAGα-HA (50 ug) representsthe baseline T cell response. Overlapping peptides spanning the entireHanoi 2005 HA protein were pooled (10 peptides/pool) and used torestimulate the splenocytes. Splenocytes were harvested 10 daysfollowing vaccination and re-stimulated using peptide pools derived fromHA. The data represents the frequency of spots per one millionsplenocytes. 4 mice were analyzed per group. From the data it can beseen that not all the hydrophobic peptides produced an equal immuneresponse.

FIG. 2: Neutralizing antibody response following immunization was doneto detect the presence of antibodies in sera that would be expected tocounter the infection. BALB/c mice were vaccinated I.M. with 50 μg ofpCAGα-HA DNA vaccine containing either: 5mer1 (SEQ ID No. 1), 5mer2 (SEQID No. 2), 5mer3 (SEQ ID No. 3), 5mer4 (SEQ ID No. 4), 5mer5 (SEQ ID No.5), 9mer1 (SEQ ID No. 6), 9mer3 (SEQ ID No. 7), 9mer4 (SEQ ID No. 8),13mer1 (SEQ ID No. 9), 13mer3 (SEQ ID No. 10), or 13mer4 (SEQ ID No. 11)foreign peptide and HA. Sera collected from immunized mice wereevaluated by neutralization assays. For neutralization assays, sera weretreated overnight at 37° C. with the receptor destroying enzyme (RDE)and then inactivated at 56° C. for 45 minutes. Two-fold serial dilutionsof each sample, starting with a 1:10 dilution, were prepared in virusdiluent and mixed with equal volume of the homologous influenza virusisolate used for immunization (100 plaque forming units [PFU] per well)and incubated at 37° C. for 60 minutes. The mixture was then transferredonto subconfluent MDCK cells in 96-well flat-bottomed plates andincubated for 5-10 minutes at room temperature. Control wells wereinfected with equal amount of viral vector without addition of serum orwith non-immune control serum. 100 μl of virus diluent supplemented with2.0 μg/ml TPCK-trypsin was then added to each well and plates wereincubated at 37° C., 5% CO₂ for 48 hr. Cells were subsequently scoredfor the presence or the absence of cytopathic effects (CPE) under alight microscope. The highest serum dilution not exhibiting CPE wasscored positive for neutralizing antibody and neutralization titers werereported as the reciprocal of this dilution. The peptides that generateda high T-cell response also generated a high neutralizing antibodyresponse.

FIG. 3: Immune cell response and protective efficacy followingimmunization with foreign peptide recombinant DNA vaccine. BALB/c micewere vaccinated I.M. with 50 μg per mouse with pCAGα-HA DNA vaccinecontaining either 5mer4 (KWCEC (SEQ ID No. 4)) or 5mer7 (KYMCW (SEQ IDNo. 12)) foreign peptide fused to the carboxyl-terminal end of HA. 5mer4(SEQ ID No. 4) was selected because of the high T-cell and neutralizingantibody response. 5mer7 (SEQ ID No. 12) was selected due to thesequence similarity to 5mer4. Hemagglutination Inhibition. Serum wascollected on day 25 post-vaccination. Serial dilutions were performed onsera obtained from vaccinated BALB/c mice and four agglutinating dosesof virus were added to each well. The sera and virus were incubated withturkey red blood cells and hemagglutination inhibition (HI titer) wasreported as the reciprocal of the highest dilution of serum which didnot block the agglutination of erythrocytes. Control mice werevaccinated with phosphate buffered saline (PBS). Error bars representthe standard deviation of the data. From the data it was surprising tosee that similar peptides behaved differently in the assay. Thereforethe sequence of the peptide is important.

FIG. 4: Immune cell response and protective efficacy followingimmunization with foreign peptide recombinant DNA vaccine. BALB/c micewere vaccinated I.M. with 50 μg per mouse with pCAGα-HA DNA vaccinecontaining either 5mer4 (SEQ ID No. 4) or 5mer7 (SEQ ID No. 12) foreignpeptide fused to the carboxyl-terminal end of HA. Neutralizing antibody(NAB) titers for pCAGα-HA-5mer4 or pCAGα-HA-5mer7 vaccinated mice. Theexperiment was repeated 3 times. Groups of 10 BALB/c mice werevaccinated I.M. with a single dose of either 1 μg pCAGα-HA-5mer4, 1 μgpCAGα-HA-5mer7, 1 μg pCAGα-HA, 5 μg pCAGα-HA or 10 μg pCAGα-HA permouse. 28 days later they were challenged with 100 LD50 of H5N1A\Hanoi\30408\2005. Control mice were vaccinated with phosphate bufferedsaline (PBS). Error bars represent the standard deviation of the data.This shows that in the response raised is one that is effective atgenerating antibodies that can effectively neutralize virus.

FIG. 5: Immune cell response and protective efficacy followingimmunization with foreign peptide recombinant DNA vaccine. BALB/c micewere vaccinated I.M. with 50 μg per mouse with pCAGα-HA DNA vaccinecontaining either 5mer4 (SEQ ID No. 4) or 5mer7 (SEQ ID No. 12) foreignpeptide fused to the carboxyl-terminal end of HA. 28 days later theywere challenged with 100 LD50 of H5N1 A\Hanoi\30408\2005 and percentsurvival over time measured. Control mice were vaccinated with phosphatebuffered saline (PBS). Error bars represent the standard deviation ofthe data. This data demonstrates that vaccination using peptides asadjuvants provided a greater level of protection than antigen alone.There is also a dose response observed with the adjuvant and that theadjuvant can result in a stronger immune response with lower levels ofantigen

FIG. 6: Immune cell response and protective efficacy followingimmunization with foreign peptide recombinant DNA vaccine. BALB/c micewere vaccinated I.M. with 50 μg per mouse with pCAGα-HA DNA vaccinecontaining either 5mer4 (SEQ ID No. 4) or 5mer7 (SEQ ID No. 12) foreignpeptide fused to the carboxyl-terminal end of HA. 28 days later theywere challenged with 100 LD50 of H5N1 A\Hanoi\30408\2005 and weightmeasured. Control mice were vaccinated with phosphate buffered saline(PBS). Error bars represent the standard deviation of the data. Thisdemonstrates that vaccination with the peptide-antigen combinationprotects the animals against weight loss which is a symptom of influenzainfection.

FIG. 7: Protective efficacy following immunization with a free(exogenous) foreign peptide for homologous challenge. Groups of 10BALB/c mice were vaccinated I.M. with a single dose of either 1 μgpCAGα-HA+5 μg 5mer4 (SEQ ID No. 4) as a free peptide, 1 μg pCAGα-HA+50μg 5mer4 (SEQ ID No. 4) as a free peptide or 1 μg pCAGα-HA+Dimethylsulfoxide (DMSO) per mouse. 28 days later they were challenged with 100LD50 of H5N1 A/Hanoi/30408/2005. Data represents percent survival.Control mice were vaccinated with PBS. This data demonstrates thatvaccination with the buffer alone does not induce protection against theinfluenza and that there is a dose effect with increasing amounts ofpeptide adjuvant.

FIG. 8: Protective efficacy following immunization with a free(exogenous) foreign peptide for homologous challenge. Groups of 10BALB/c mice were vaccinated I.M. with a single dose of either 1 μgpCAGα-HA+5 μg 5mer4 (SEQ ID No. 4) as a free peptide, 1 μg pCAGα-HA+50μg 5mer4 (SEQ ID No. 4) as a free peptide or 1 μg pCAGα-HA+Dimethylsulfoxide (DMSO) per mouse. 28 days later they were challenged with 100LD50 of H5N1 A/Hanoi/30408/2005. Data represents body weight over time.Control mice were vaccinated with PBS. This demonstrates thatvaccination with the peptide plus antigen protects the animals againstweight loss which is a symptom of influenza infection.

FIG. 9: Protective efficacy following immunization with a free(exogenous) foreign peptide for homologous challenge. Groups of 10BALB/c mice were vaccinated I.M. with a single dose of either 1 μgpCAGα-HA+5 μg 5mer4 (SEQ ID No. 4) as a free peptide, 1 μg pCAGα-HA+50μg 5mer4 (SEQ ID No. 4) as a free peptide or 1 μg pCAGα-HA+Dimethylsulfoxide (DMSO) per mouse. 28 days later they were challenged with 100LD50 of H5N1 A/Hanoi/30408/2005. Data represents percent survival.Control mice were vaccinated with PBS. This data demonstrates thatvaccination with the buffer alone does not induce protection against theinfluenza and that there is a dose effect with increasing amounts ofpeptide adjuvant.

FIG. 10: Protective efficacy following immunization with different dosesof free peptide 5mer4 (SEQ ID No. 4). Groups of 10 BALB/c mice werevaccinated I.M. with a single dose of 100 μg pCAGα-HA+ either 50 μg5mer4 (SEQ ID No. 4) or 100 μg 5mer4 (SEQ ID No. 4) as a free peptideper mouse. 28 days later they were challenged with 100 LD50 ofhomologous Hanoi 2005 virus. Data represents body weight over time.Control mice were vaccinated with PBS or 50 ug of the free peptide. Thisdemonstrates that vaccination with the peptide plus Antigen protects theanimals against weight loss which is a symptom of influenza infection.

FIG. 11: Protective efficacy following immunization with different dosesof free peptide 5mer4. Groups of 10 BALB/c mice were vaccinated I.M.with a single dose of 100 μg pCAGα-HA+ either 50 μg 5mer4 (SEQ ID No. 4)or 100 μg 5mer4 (SEQ ID No. 4) as a free peptide per mouse. 28 dayslater they were challenged with 100 LD50 of homologous Hanoi 2005 virus.Data represents percent survival. Control mice were vaccinated with PBSor 50 ug of the free peptide. This demonstrates that the effect requiresboth antigen and adjuvant to be effective in raising a specific immuneresponse.

FIG. 12: Protective efficacy following immunization with a free foreignpeptide for heterologous challenge. Groups of 10 BALB/c mice werevaccinated I.M. with a single dose of 100 μg pCAGα-HA+50 μg 5mer4 (SEQID No. 4) as a free peptide per mouse. 28 days later they werechallenged with 100 LD50 of mouse-adapted H5N1 A\Hong Kong\483\1997.Data represents percent body weight loss over time. Control mice werevaccinated with PBS. This demonstrates that vaccination with the peptideplus antigen protects the animals against weight loss which is a symptomof influenza infection.

FIG. 13: Protective efficacy following immunization with a free foreignpeptide for heterologous challenge. Groups of 10 BALB/c mice werevaccinated I.M. with a single dose of 100 μg pCAGα-HA+50 μg 5mer4 (SEQID No. 4) as a free peptide per mouse. 28 days later they werechallenged with 100 LD50 of mouse-adapted H5N1 A\Hong Kong\483\1997.Data represents percent survival. Control mice were vaccinated with PBS.This demonstrates the efficacy of the vaccination procedure againstheterologous viruses.

FIG. 14: Enzyme-linked immunosorbent assay (ELISA) following vaccinationwith the Hepatitis B vaccine (Engerix-B). BALB/c mice were vaccinatedI.M. with the equivalent of 1 ug of the Engerix-B vaccine with (A) 50 ugof free peptide or (B) without peptide as a control. Serum was obtainedfrom the mice at weeks 2, 4, 6, and 8 following vaccination. Totalanti-HBS antibodies were detected using a commercial ELISA kit. Thisdata indicates a much stronger immune response is generated in thepresence of antigen plus peptide than with antigen alone. The timecourse of immune response is as expected.

FIG. 15: Enzyme-linked immunosorbent assay (ELISA) following vaccinationwith the seasonal influenza (Fluviral 2008-2009) vaccine. BALB/c micewere vaccinated I.M. with the equivalent of 5 ug of the Fluviral vaccinewith (A) 50 ug of free peptide or (B) without peptide as a control.Serum was obtained from the mice at weeks 2, 4, 6, and 8 followingvaccination. Total anti-influenza IgG antibodies were detected using acommercial ELISA kit. This data demonstrates that even in the presenceof high levels of antigen, the peptide will boost immune response.

FIG. 16: Enzyme-linked immunosorbent spot (ELISPOT) T cell responsefollowing immunization. BALB/c mice were vaccinated I.M. with 50 μg ofpCAGα-HA DNA vaccine+50 ug of 5mer4 (SEQ ID No. 4), CpG-ODN (10 μg),alum (Alhydrogel, 450 ug), a combination of all three, or HA alone.Splenocytes were harvested 10 days following vaccination andre-stimulated using peptide pools derived from HA. The data representsthe frequency of spots per one million splenocytes. 4 mice were analyzedper group. This data shows that the immune response generated when usingthe peptide is stronger than that generated with alum alone andcomparable to that generated by CpG. Also, there is an apparent additiveeffect when the adjuvants are combined.

FIG. 17: Enzyme-linked immunosorbent spot (ELISPOT) T cell responsefollowing immunization. BALB/c mice were vaccinated I.M. with 50 μg ofpCAGα-HA DNA vaccine alone or combined with pools of 10 5mer peptides.Splenocytes were harvested 10 days following vaccination andre-stimulated using peptide pools derived from HA. The data representsthe frequency of spots per one million splenocytes. Bars represent thetotal of all pools. 4 mice were analyzed per group. This approachenabled rapid screening of closely related peptides.

FIG. 18: Enzyme-linked immunosorbent spot (ELISPOT) T cell responsefollowing immunization. BALB/c mice were vaccinated I.M. with 50 μg ofpCAGα-HA DNA vaccine alone or combined with 50 ug of a 5mer from animmunodominant pool. Splenocytes were harvested 10 days followingvaccination and re-stimulated using peptide pools derived from HA. Thedata represents the frequency of spots per one million splenocytes. Barsrepresent the total of all pools. 4 mice were analyzed per group. Thisdata shows that despite single amino acid changes in peptide sequence,there can be a large difference in the strength of the T-cell responsethat is generated.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. All publications mentioned hereunderare incorporated herein by reference.

Adjuvant compositions comprising specific 5mer polypeptides incombination with antigen delivery systems and/or immunostimulatorymolecules, such as immunostimulatory nucleic acid sequences, forenhancing the immune response of a coadministered antigen, aredescribed. The present invention is based in part on the surprisingdiscovery that the use selected hydrophobic peptides in combination withantigens provides for significantly higher antibody titers to acoadministered antigen, than those observed without such deliverysystems or using traditional adjuvants. The use of such combinationsprovides a safe and effective approach for enhancing the immunogenicityof a variety of vaccine antigens for use in both prophylactic andtherapeutic compositions.

The practice of the present invention will employ, unless otherwiseindicated, conventional methods of chemistry, biochemistry, recombinantDNA techniques and immunology, within the skill of the art. Suchtechniques are explained fully in the literature. See, e.g., FundamentalVirology, 2nd Edition, vol. I & II (B. N. Fields and D. M. Knipe, eds.);Handbook of Experimental Immunology, Vols. I-IV (D. M. Weir and C. C.Blackwell eds., Blackwell Scientific Publications); T. E. Creighton,Proteins: Structures and Molecular Properties (W. H. Freeman andCompany, 1993); A. L. Lehninger, Biochemistry (Worth Publishers, Inc.,current addition); Sambrook, et al., Molecular Cloning: A LaboratoryManual (2nd Edition, 1989); Methods In Enzymology (S. Colowick and N.Kaplan eds., Academic Press, Inc.).

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entirety.

It must be noted that, as used in this specification and the appendedclaims, the singular forms “a”, “an” and “the” include plural referentsunless the content clearly dictates otherwise. Thus, for example,reference to “an antigen” includes a mixture of two or more antigens,and the like.

The following amino acid abbreviations are used throughout the text:

Alanine: Ala (A) Arginine: Arg (R) Asparagine: Asn (N) Aspartic acid:Asp (D) Cysteine: Cys (C) Glutamine: Gln (Q) Glutamic acid: Glu (E)Glycine: Gly (G) Histidine: His (H) Isoleucine: Ile (I) Leucine: Leu (L)Lysine: Lys (K) Methionine: Met (M) Phenylalanine: Phe (F) Praline: Pro(P) Serine: Ser (S) Threonine: Thr (T) Tryptophan: Trp (W) Tyrosine: Tyr(Y) Valine: Val (V)

DEFINITIONS

In describing the present invention, the following terms will beemployed, and are intended to be defined as indicated below.

The terms “polypeptide” and “protein” refer to a polymer of amino acidresidues and are not limited to a minimum length of the product. Thus,peptides, oligopeptides, dimers, multimers, and the like, are includedwithin the definition. Both full-length proteins and fragments thereofare encompassed by the definition. The terms also include postexpressionmodifications of the polypeptide, for example, glycosylation,acetylation, phosphorylation and the like. Furthermore, for purposes ofthe present invention, a “polypeptide” refers to a protein whichincludes modifications, such as deletions, additions and substitutions(generally conservative in nature), to the native sequence, so long asthe protein maintains the desired activity. These modifications may bedeliberate, as through site-directed mutagenesis, or may be accidental,such as through mutations of hosts which produce the proteins or errorsdue to PCR amplification.

By “antigen” is meant a molecule, which contains one or more epitopesthat will stimulate a host's immune system to make a cellularantigen-specific immune response when the antigen is presented, or ahumoral antibody response. The term “antigen” as used herein denotesboth subunit antigens, i.e., proteins which are separate and discretefrom a whole organism with which the antigen is associated in nature, aswell as killed, attenuated or inactivated bacteria, viruses, parasitesor other microbes. Antibodies such as anti-idiotype antibodies, orfragments thereof, and synthetic peptide mimotopes, which can mimic anantigen or antigenic determinant, are also captured under the definitionof antigen as used herein. Similarly, an oligonucleotide orpolynucleotide which expresses a therapeutic or immunogenic protein, orantigenic determinant in vivo, such as in gene therapy and nucleic acidimmunization applications, is also included in the definition of antigenherein. Further, for purposes of the present invention, antigens can bederived from any of several known viruses, bacteria, parasites andfungi, as well as any of the various tumor antigens.

An “immunological response” to a selected antigen or composition is thedevelopment in a subject of a humoral and/or a cellular immune responseto molecules present in the composition of interest. For purposes of thepresent invention, a “humoral immune response” refers to an immuneresponse mediated by antibody molecules, while a “cellular immuneresponse” is one mediated by T-lymphocytes and/or other white bloodcells. One important aspect of cellular immunity involves anantigen-specific response by cytolytic T-cells (“CTLs”). CTLs havespecificity for peptide antigens that are presented in association withproteins encoded by the major histocompatibility complex (MHC) andexpressed on the surfaces of cells. CTLs help induce and promote theintracellular destruction of intracellular microbes, or the lysis ofcells infected with such microbes. Another aspect of cellular immunityinvolves an antigen-specific response by helper T-cells. Helper T-cellsact to help stimulate the function, and focus the activity of,nonspecific effector cells against cells displaying peptide antigens inassociation with MHC molecules on their surface. A “cellular immuneresponse” also refers to the production of cytokines, chemokines andother such molecules produced by activated T-cells and/or other whiteblood cells, including those derived from CD4+ and CD8+ T-cells. Acomposition or vaccine that elicits a cellular immune response may serveto sensitize a vertebrate subject by the presentation of antigen inassociation with MHC molecules at the cell surface. The cell-mediatedimmune response is directed at, or near, cells presenting antigen attheir surface. In addition, antigen-specific T-lymphocytes can begenerated to allow for the future protection of an immunized host. Theability of a particular antigen to stimulate a cell-mediatedimmunological response may be determined by a number of assays, such asby lymphoproliferation (lymphocyte activation) assays, CTL cytotoxiccell assays, or by assaying for T-lymphocytes specific for the antigenin a sensitized subject. Such assays are well known in the art. See,e.g., Erickson et al., J. Immunol. (1993) 151:4189-4199; Doe et al.,Eur. J. Immunol. (1994) 24:2369-2376.

The terms “effective amount” or “pharmaceutically effective amount” ofan adjuvant composition and antigen, as provided herein, refer to anontoxic but sufficient amount of the composition to provide the desiredresponse, such as an immunological response, and optionally, acorresponding therapeutic effect, or in the case of delivery of atherapeutic protein, an amount sufficient to effect treatment of thesubject, as defined below. As will be pointed out below, the exactamount required will vary from subject to subject, depending on thespecies, age, and general condition of the subject, the severity of thecondition being treated, and the particular macromolecule of interest,mode of administration, and the like. An appropriate “effective” amountin any individual case may be determined by one of ordinary skill in theart using routine experimentation.

The “antigen delivery system” comprises the adjuvant composition, andantigen and other buffers and substances which may be used to stabilizeor act as carriers for the combination.

In a first embodiment, the peptides are used in conjunction withantigens to generate a humoral and cellular response aimed to prevent aninfectious disease by co-administering a peptide with an antigen.

In yet another embodiment, the subject invention is directed to a methodof stimulating an immune response in a vertebrate subject whichcomprises administering to the subject an effective amount or atherapeutically effective amount of a selected antigen and an adjuvantcomposition comprising a peptide as described herein. As will beappreciated by one of skill in the art, the antigen and the adjuvantpeptide may be administered by a variety of means and under a variety ofconditions within the invention. For example, there may be provided anantigen delivery system and/or an immunostimulatory molecule, whereinthe adjuvant composition is capable of increasing the immune response tothe selected antigen. The antigen may be present in the adjuvantcomposition or may be administered in a separate composition. If theantigen is delivered separately, it may be delivered to the same ordifferent site, and may be delivered prior to, subsequent to, orconcurrent with the adjuvant composition.

It is important to note that as described herein, the administration ofthe adjuvant peptide to the individual in need of or desirous of astimulated immune response for example to an antigen may be done by avariety of means, for example, by administering the adjuvant peptide andthe antigen together, separately or even at different locations asdiscussed herein and as known in the art. The adjuvant peptide may beadministered as a purified or isolated peptide or may be fused to theantigen either chemically or genetically (i.e. a transgenic peptidecomprising both a peptide antigen and the adjuvant peptide) or it may beadministered as a nucleic acid comprising the adjuvant peptide which isarranged to be expressed following administration so that the adjuvantpeptide is still administered to the individual.

In a related embodiment, the subject invention is directed to a methodof preventing infectious disease by co-administering a selected peptideand one or more DNA sequences that can express protein(s) from theinfectious agent. These agents could include viruses such as HepatitisC, HIV, hemoragghic fevers and the like or other antigens where a strongT-cell response is desired. As will be appreciated by one of skill inthe art, suitable agents for co-administration include but are by nomeans limited to DNA, RNA or protein vaccines, viral extracts anddeactived viruses or bacteria.

In one aspect of the invention, there is provided a compositioncomprising an effective amount of an adjuvant peptide comprising anamino acid sequence selected from the group consisting of: KWCEC (SEQ IDNo. 4); KYMCW (SEQ ID No. 12); CYWWW (SEQ ID No. 14); EHWCM (SEQ ID No.15); FCCWW (SEQ ID No. 16); TCCMW (SEQ ID No. 17); TCWWH (SEQ ID No.18); TCYWW (SEQ ID No. 19); WMICM (SEQ ID No. 20) and YWHMW (SEQ ID No.21) and an antigen of interest.

In another aspect of the invention, there is provided a method ofstimulating an immune response or enhancing an immune response to anantigen comprising administering to an individual in need of or desirousof such treatment an effective amount of an adjuvant peptide comprisingan amino acid sequence selected from the group consisting of: KWCEC (SEQID No. 4); KYMCW (SEQ ID No. 12); CYWWW (SEQ ID No. 14); EHWCM (SEQ IDNo. 15); FCCWW (SEQ ID No. 16); TCCMW (SEQ ID No. 17); TCWWH (SEQ ID No.18); TCYWW (SEQ ID No. 19); WMICM (SEQ ID No. 20) and YWHMW (SEQ ID No.21). The individual in need of or desirous of such treatment may be anindividual who is being immunized, as discussed herein.

In one preferred embodiment, the adjuvant peptide is selected from thegroup consisting of KWCEC (SEQ ID No. 4); KYMCW (SEQ ID No. 12); TCCMW(SEQ ID No. 17); TCWWH (SEQ ID No. 18); and TCYWW (SEQ ID No. 19).

In another preferred embodiment, the adjuvant peptide is selected fromthe group consisting of FCCWW (SEQ ID No. 16); TCCMW (SEQ ID No. 17);TCWWH (SEQ ID No. 18); TCYWW (SEQ ID No. 19); and WMICM (SEQ ID No. 20)

In another preferred embodiment, the adjuvant peptide is selected fromthe group consisting of KWCEC (SEQ ID No. 4) and KYMCW (SEQ ID No. 12).

In another preferred embodiment, the adjuvant peptide is KWCEC (SEQ IDNo. 4).

In a preferred embodiment, the ‘effective amount’ or ‘therapeuticallyeffective amount’ of the adjuvant peptide is between about 50 μg andabout 5 mg per dose or per administration. In a more preferredembodiment, the dosage is between about 50 μg and about 500 μg. As willbe appreciated by one of skill in the art, the effective amount may varyaccording to the age, weight and condition of the individual to which itis being administered.

As discussed herein, the adjuvant peptide of the invention may beadministered as ‘free’ peptides (that is, may be isolated peptidesconsisting of an amino acid sequence selected from the group consistingof: KWCEC (SEQ ID No. 4); KYMCW (SEQ ID No. 12); CYWWW (SEQ ID No. 14);EHWCM (SEQ ID No. 15); FCCWW (SEQ ID No. 16); TCCMW (SEQ ID No. 17);TCWWH (SEQ ID No. 18); TCYWW (SEQ ID No. 19); WMICM (SEQ ID No. 20) andYWHMW (SEQ ID No. 21). Alternatively, the amino acid sequence may beattached to or embedded within an antigenic peptide or a carrier peptideusing means known in the art. In other embodiments, such a construct maybe encoded by a nucleic acid molecule which may be administered to theindividual such that the adjuvant peptide is expressed followingadministration as discussed herein.

As will be appreciated by one of skill in the art, any suitable antigenmay be used in combination with the adjuvant peptide of the invention.In a particularly preferred embodiment, the antigen is an antigen froman infectious disease, for example, a deactivated or attenuated virus orbacterium, a viral or bacterial extract or a bacterial or viral peptide.

In another aspect of the invention, there is provided the use of theabove-described adjuvant peptides for inducing or stimulating orenhancing an immune response in an individual in need of such treatment.As discussed above, the adjuvant peptides may be administered togetherwith the antigen or may be administered separately or may beadministered at different sites.

In another aspect of the invention, there is provided a method ofpreparing a medicament or composition for stimulating an immune responsein an individual comprising mixing an adjuvant peptide as describedherein with a suitable excipient, for example, a suitable vaccineexcipient, carrier or diluent. In other embodiments, the medicament orcomposition or vaccine may be prepared by mixing the adjuvant peptide asdescribed above with the desired antigen.

As discussed herein, the adjuvant peptides may be administered to anyvertebrate but preferably are administered to humans or animals forexample in veterinary applications. Accordingly, in some aspects of theinvention, the ‘individual’ is a non-human animal or a non-humanvertebrate animal. Alternatively, the adjuvant peptides may be used forresearch purposes.

EXAMPLES

Below are examples of specific embodiments for carrying out the presentinvention. The examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

Example 1 Identification of Peptides

Proteome databases were screened using a computer algorithm looking forshort 5-mers peptides of amino acids sequences occurring maximum once.This analysis generated 417 never observed and 1288 unique sequences of5-mers peptide found only once in all known proteomes. Nine andthirteen-mers sequences were computer generated from the previously 417identified 5-mers sequences absent from known proteomes. Six 5-mers,three 9-mers and three 13-mers of various predicted hydrophobicityvalues were randomly selected for functional analysis. The effect ofeach short peptide on the immune response was first analyzed byevaluating the T-cell response against the hemagglutinin (HA) antigen ofthe Hanoi 2005 avian influenza virus expressed from a pCAG-based DNAvaccine in Balb/c mice. Each 5-, 9- and 13-mers sequence was cloned inframe at the C-terminus of the HA antigen in order to facilitateexpression and minimize potential experimental deviation originatingfrom independent peptidic preparations of variable purity. Groups of 4mice were vaccinated intramuscularly (I.M.) with 50 μg per mouse of eachplasmid DNA encoding for HA in frame with each short peptide sequenceand the T-cell response was monitored from splenocytes 10 days later.The same plasmid DNA encoding HA without additional sequences (pCAG-HA)was included as a benchmark control. A library of overlapping peptidescovering the entire HA protein was used for re-stimulating splenocytesand IFN-g production was evaluated by ELISPOT as a measure of the T-cellresponse. FIG. 1 shows that pCAG-HA-5-mers #4 (SEQ ID No. 4) and #6(DMCKW, SEQ ID No. 13) increased the IFN-g production followingstimulation with several individual peptides from the HA library whencompared to other modified pCAG-HA-5, 9 or 13-mers or with theunmodified pCAG-HA control. From the data, it can be concluded that5mers worked better than 9mers or 13mers at generating a T-cellresponse. From the data is does not appear that there are any patternsregarding hydrophobicity or sequence that led to an increased T-cellresponse which was quite surprising. The hydrophobicities of the variouspeptides are listed in Table 1.

In addition neutralizing antibodies to the HA virus were measured and5mer4 and 5mer7 were identified as peptides that caused a significantincrease in neutralizing antibody response. Additional peptides wereidentified using a similar process. (see FIG. 17 and FIG. 18 It wassurprising to find that very similar peptides could have a dramaticallydifferent effect on the observed response as shown in FIG. 18 (sequencesshown in Table 2). Neutralizing antibodies are a marker for efficacy andit appears that vaccination with the 5mer4 (SEQ ID No. 4) and 5mer7 (SEQID No. 12) as adjuvants caused a significant antibody response whileother 5mers (5mer1 (SEQ ID No. 1), 5mer2 (SEQ ID No. 2), 5mer3 (SEQ IDNo. 3)) and the 9 and 13mers did not cause a strong neutralizingantibody response as shown in FIG. 2. Therefore to search for additionalpeptides that might also cause an increased immune response, groups of10 randomly selected peptides were screened in the T-cell assaydescribed above. For groups that demonstrated a T-cell response abovebaseline, individual peptides were screened in the assay. From the datait is surprising that closely related sequences had vastly differenteffects on the generation of a T-cell response. For example, peptidesequences CYWWW (91, SEQ ID No. 14) generated a significant T-cellresponse, but the peptide CYYWC (92, SEQ ID No. 22) which is differentby only one amino acid generated a T-cell response that was below thebaseline of HA alone. Similar differences were found for other peptidessuch the pair of EHWCM (93, SEQ ID No. 15)) and EMWCM (94, SEQ ID No.23)) where the former generated a large T-cell response, but the latterdid not. Peptides that generated a strong T-cell response in this assayare predicted to be good adjuvants based on the responses generated by5mer4 (KWCEC, SEQ ID No. 4)) and 5mer7 (KYMCW (SEQ ID No. 12)) inexpanded animal studies. It can also be observed from the data that thepeptides that generated a high T-cell response also generated a highneutralizing antibody response which would be predicted to lead to anefficacious response in survival studies.

The anticipated dose of the peptide adjuvant in humans is expected to bebetween 50 μg and 5 mg, depending on the nature of the antigen. Mostadjuvants are used at between 50 μg and 500 μg and this is expected tohold true for the selected peptides as well. To date, no serioustoxicity has been observed with high doses of the selected peptides.

Example 2 Generation of a Protective Immune Response After Vaccination

Based on induced higher T-cell responses, pCAG-HA fused to either 5mer4(SEQ ID No. 4) or 5mer7 (SEQ ID No. 12) were further studied in Balb/cmice. The antibody response was monitored by hemagglutination inhibition(HI) and neutralization (NAB) titration assays of sera 25 days afterI.M. vaccination with each DNA vaccine including the unmodified HA as acontrol. The average HI reciprocal dilution titer was 85±40, 55±35 or25±20 while the NAB titer was 25±30, 22±10 or undetectable forpCAG-HA-5-mer4, and 5mer7 or pCAG-HA respectively. To assess whetherhigher T and B-cell responses would correlate with enhanced protection,Balb/c mice were challenged with a lethal dose of Hanoi05 28 days afterI.M. immunization with each HA-5mer4 and 5mer7. The DNA vaccine doseselected was based on the dose of 1 μg of unmodified pCAG-HA which wasfound to be the minimal dose tested to induce survival with 30%.Vaccination with 1 μg of pCAG-HA-5mer7 protected 80% of the animals fromdeath with a weight loss of 5% while pCAG-HA-5-mers #4 induced 100%survival with no statistically significant weight loss and no clinicalsigns of disease. This demonstrates that using the peptide attached tothe antigen as an adjuvant enables an effective immune response to begenerated and provides protection from the effects typically found oninfluenza infection.

Example 3 Generation of a Protective Immune Response After Vaccination

Based on induced higher T-cell responses, pCAG-HA combined with 5 or 50μg free peptides 5mer4 (SEQ ID No. 4) were further studied in Balb/cmice. The antibody response was monitored by hemagglutination inhibition(HI) and neutralization (NAB) titration assays of sera 25 days afterI.M. vaccination with each DNA vaccine including the unmodified HA as acontrol. To assess whether higher T and B-cell responses would correlatewith enhanced protection, Balb/c mice were challenged with a lethal doseof Hanoi05 28 days after I.M. immunization with each HA plus 5 or 50 μgof 5mer4 (SEQ ID No. 4). The DNA vaccine dose selected was based on thedose of 1 μg of unmodified pCAG-HA which was found to be the minimaldose tested to induce survival with 30%. Vaccination with 1 μg ofpCAG-HA plus 5 ug 5mer4 (SEQ ID No. 4) protected 50% of the animals fromdeath with minimal weight loss of while pCAG-HA plus 50 μg of 5mer4induced 90% survival with no statistically significant weight loss andno clinical signs of disease. The unvaccinated control animals treatedhad 100% mortality. This demonstrates that using the free peptide as anadjuvant in conjunction with the antigen enables an effective immuneresponse to be generated and provides protection from the effectstypically found on influenza infection. This data also demonstrates thatthere is a dose response with different levels of adjuvant.

Example 4 Protective Efficacy Following Immunization with a Free ForeignPeptide for Heterologous Challenge

Groups of 10 BALB/c mice were vaccinated I.M. with a single dose of 100μg pCAGα-HA+ either 50 or 100 μg 5mer4 (SEQ ID No. 4) as a free peptideper mouse. Control mice were immunized with only 50 μg of 5mer4 or PBS.28 days later they were challenged with 100 LD50 of mouse-adapted H5N1A\Hong Kong\483\1997. the groups of mice that were immunized with pCAGα-HA+ either 50 or 100 μg 5mer4 (SEQ ID No. 4) both showed 100% survivalwhile the control PBS group showed 100% mortality at 18 days postchallenge. This demonstrates that using the free peptide as an adjuvantalong with an antigen enables an effective immune response to begenerated and provides protection from the effects typically found oninfluenza infection using related viruses which suggests that a strong,broad cross protective immune response has been generated.

Example 5 Adjuvant Works with Multiple Antigens

To demonstrate that the effect is not limited to influenza vaccines,BALB/c mice were vaccinated I.M. with the equivalent of 1 ug of theHepatitis B vaccine (Engerix-B) Engerix-B vaccine with 50 μg of freepeptide or without peptide as a control. Serum was obtained from themice at weeks 2, 4, 6, and 8 following vaccination. Total anti-FIBSantibodies were detected using a commercial ELISA kit. The response rateis quite dramatic, with the control Engerix B group showing a very smallresponse at 8 weeks post immunization, and the Engerix B plus 50 μg ofpeptide showing a strong response at both 6 and 8 weeks postimmunization. The generation of an antibody response at 6-8 weeks postimmunization is typical of the type of response generated withvaccinations. This demonstrates that the free peptide functions with awide range of antigen types.

Example 6 Adjuvant Works with Multiple Antigens

To demonstrate that the effect is not limited to influenza vaccines,BALB/c mice were vaccinated I.M. with the equivalent of 5 μg of thefluviral vaccine. Fluviral vaccine with (A) 50 ug of free peptide or (B)without peptide as a control. Serum was obtained from the mice at weeks2, 4, 6, and 8 following vaccination. Total anti-HBS antibodies weredetected using a commercial ELISA kit. The response rate is quitedramatic, with the control fluviral group showing a small response at 8weeks post immunization, and the fluviral plus 50 μg of peptide showinga stronger response at both 6 and 8 weeks post immunization. Since thisstudy was done with a relatively high level of antigen, it suggests thatthe amount of antigen required might be lower than typically expected togenerate a strong immune response. This also demonstrates that the freepeptide functions with a wide range of antigen types.

While the preferred embodiments of the invention have been describedabove, it will be recognized and understood that various modificationsmay be made therein, and the appended claims are intended to cover allsuch modifications which may fall within the spirit and scope of theinvention.

TABLE 1 Selected sequences Amino Molecu- Acid lar SequenceHydrophobicity Weight Name KWCEC 0.74* 649.78 5 mer4 SEQ ID No. 4 KYMCW1.00* 711.90 5 mer7 SEQ ID No. 12 CYWWW 1.85  824.95 SEQ ID No. 14 EHWCM0.90  686.81 SEQ ID No. 15 FCCWW 1.87* 725.88 SEQ ID No. 16 TCCMW 1.36*624.80 SEQ ID No. 17 TCWWH 1.29* 713.81 SEQ ID No. 18 TCYWW 1.45* 739.85SEQ ID No. 19 WMICM 1.80  682.92 SEQ ID No. 20 YWHMW 1.36  803.94SEQ ID No. 21 Sequences of 5 mers 91-99 (from FIG. 18) Sequence NumberSequence 91 (SEQ ID No. 14) CYWWW 92 (SEQ ID No. 22) CYYWC93 (SEQ ID No. 15) EHWCM 94 (SEQ ID No. 23) EMWCM 95 (SEQ ID No. 24)EWCMC 96 (SEQ ID No. 25) EWNCW 97 (SEQ ID No. 26) EYCWW98 (SEQ ID No. 16) FCCWW 99 (SEQ ID No. 27) FHMMW

The invention claimed is:
 1. A composition comprising an effectiveamount of an adjuvant peptide comprising the amino acid sequence as setforth in SEQ ID NO:4 and an antigen of interest.
 2. The compositionaccording to claim 1 wherein the effective amount of the adjuvantpeptide is between about 50 μg and about 5 mg.
 3. The compositionaccording to claim 1 wherein the effective amount of the adjuvantpeptide is between about 50 μg and about 500 μg.
 4. A method ofstimulating an immune response or enhancing an immune response to anantigen comprising administering to an individual in need of or desirousof such treatment an effective amount of an adjuvant peptide comprisingthe amino acid sequence as set forth in SEQ ID NO:4.
 5. The methodaccording to claim 4 wherein the effective amount is between about 50 μgand about 5 mg.
 6. The method according to claim 4 wherein the effectiveamount is between about 50 μg and about 500 μg.
 7. The method accordingto claim 4 wherein the individual is a non-human animal.
 8. A method ofpreparing a medicament for stimulating an immune response or enhancingan immune response to an antigen comprising mixing an effective amountof an adjuvant peptide comprising the amino acid sequence as set forthin SEQ ID NO:4 with a suitable excipient.
 9. The method according toclaim 8 wherein the effective amount is between about 50 μg and about 5mg.
 10. The method according to claim 8 wherein the effective amount isbetween about 50 μg and about 500 μg.